Significance

Many animals use the sun or moon and the polarization pattern for navigation. We combined behavioral experiments with physiological measurements of brain activity to reveal which celestial cue dominates the orientation compass of diurnal and nocturnal dung beetles. The preference found behaviorally precisely matches the preference encoded neurally and shows how the brain dynamically controls the cue preference for orientation at different levels: The sun or moon always dominates the orientation behavior and neural tuning of diurnal beetles, whereas in nocturnal beetles, celestial bodies dominate tuning only in bright light, with a switch to polarized light at night. This flexible neural tuning in the nocturnal species provides a simple mechanism that allows it to use the most reliable available orientation cue.

Abstract

Diurnal and nocturnal African dung beetles use celestial cues, such as the sun, the moon, and the polarization pattern, to roll dung balls along straight paths across the savanna. Although nocturnal beetles move in the same manner through the same environment as their diurnal relatives, they do so when light conditions are at least 1 million-fold dimmer. Here, we show, for the first time to our knowledge, that the celestial cue preference differs between nocturnal and diurnal beetles in a manner that reflects their contrasting visual ecologies. We also demonstrate how these cue preferences are reflected in the activity of compass neurons in the brain. At night, polarized skylight is the dominant orientation cue for nocturnal beetles. However, if we coerce them to roll during the day, they instead use a celestial body (the sun) as their primary orientation cue. Diurnal beetles, however, persist in using a celestial body for their compass, day or night. Compass neurons in the central complex of diurnal beetles are tuned only to the sun, whereas the same neurons in the nocturnal species switch exclusively to polarized light at lunar light intensities. Thus, these neurons encode the preferences for particular celestial cues and alter their weighting according to ambient light conditions. This flexible encoding of celestial cue preferences relative to the prevailing visual scenery provides a simple, yet effective, mechanism for enabling visual orientation at any light intensity.

The blue sky is a rich source of visual cues that are used by many animals during orientation or navigation (1, 2). Besides the sun, celestial phenomena, such as the skylight intensity gradient or the more complex polarization pattern, can serve as references for spatial orientation (3⇓–5). Polarized skylight is generated by scattered sunlight in the atmosphere, and to a terrestrial observer, the resulting alignment of the electric field vectors extends across the entire sky, forming concentric circles around the position of the sun (Fig. 1A). A similar distribution of brightness and polarization pattern is also created around the moon (6). Although this nocturnal pattern is 1 million-fold dimmer than the daylight pattern (6), some animals, such as South African ball-rolling dung beetles, can use this lunar polarization pattern for orientation (7). To avoid competition for food at the dung pile, these beetles detach a piece of dung, shape it into a ball, and roll it away along a straight-line path. For this type of straight-line orientation, nocturnal beetles seem to rely exclusively on celestial cues (8), such as the moon or polarized light.

Celestial cue preference in dung beetles under a natural sky. (A) Schematic illustration of the polarization pattern around a celestial body (sun or moon). Change of direction in diurnal (D, Left) and nocturnal (N, Right) beetles rolling under a sun-lit (B) or moon-lit (C) sky. The change of direction was calculated as the angular difference between two consecutive rolls, either without manipulation (control, ●) or when the sun or moon was reflected to the opposite sky hemisphere between the two rolls (test, ○). The mean directions (μ) are indicated by black (control) or red (test) lines, and error bars indicate circular SDs. (B) Without manipulation, both species kept the direction [P < 0.001 by V test; μdiurnal (±SD) = 2.6° ± 17.98°, n = 20; μnocturnal = −8.7° ± 38.34°, n = 20). When the sun was reflected to the opposite sky hemisphere (and the real sun was shaded), both species responded to this change (P < 0.001 by V-test; μdiurnal = 178.9° ± 54.6°, n = 20; μnocturnal = 163.8° ± 46.58°, n = 20). (C) Under the moon in the control experiments, both species showed a constant rolling direction (P < 0.001 by V test; μdiurnal = 3.1° ± 35.39°, n = 20; μnocturnal = −3.3° ± 37.87°, n = 20). When the moon was reflected to the opposite sky hemisphere, the diurnal species followed the position change of the moon (P = 0.002 by V test; μ = 179.5° ± 72.37°, n = 20), whereas the nocturnal species continued rolling in the original rolling direction (P < 0.001 by V test; μ = −13.4° ± 74.27°, n = 20).

As with all nocturnal animals, night-active beetles have to overcome a major challenge: They need to maintain high orientation precision even under extremely dim light conditions. Indeed, recent experiments have shown that nocturnal dung beetles orient at night with the same precision as their diurnal relatives during the day (9), an ability partly due to the fact that their eyes are considerably more sensitive than the eyes of species that are active at brighter light levels (10⇓–12). Nonetheless, for each species, orientation precision relies on being tuned to the most reliable celestial compass cue that is available during the animal’s normal activity window. How salient are these cues for nocturnal and diurnal species? Do diurnal species have a different celestial cue preference than nocturnal species? If so, how are these preferences represented neurally in the brain?

In this study, we present a detailed picture of how the orientation systems of two closely related nocturnal and diurnal animals have been adapted to the ambient light conditions, combining behavioral experiments from the field with electrophysiological investigations of the underlying neural networks. Using behavioral experiments, we show that nocturnal dung beetles switch from a compass that uses a discrete celestial body (the sun) during the day to a celestial polarization compass for dim light orientation at night, whereas diurnal beetles use a celestial body (the sun or moon) for orientation at all light levels. In a second step, we simulated these skylight cues (the sun or moon and the polarization pattern) while electrophysiologically recording responses from neurons in the dung beetle’s central complex, a brain area that has been suggested to house the internal compass for celestial orientation (13, 14). These neural data precisely matched the cue preferences observed in behavioral field trials and show how an animal’s visual ecology influences the neural activity of its sky compass neurons. Our results also reveal, for the first time to our knowledge, how a weighting of celestial orientation cues could be neurally encoded in an animal brain.

Results and Discussion

Nocturnal Dung Beetles Use the Polarization Pattern as a Primary Orientation Cue at Night.

Dung beetles use a large repertoire of celestial cues for orientation (5, 7, 15⇓–17), but their two main compass cues are (i) the sun or moon and (ii) the celestial polarization pattern (5). Here, we first sought to test if one compass cue is dominant over the other in the orientation systems of two closely related dung beetle species that are active at different times of the day: Scarabaeus lamarcki (diurnal) and Scarabaeus satyrus (nocturnal). The robust straight-line orientation behavior of these beetles can be induced independent of the time of day, allowing us to examine the beetles’ choice of celestial cues even at times when they are not usually active (i.e., the nocturnal species during the day, the diurnal species at night). First, we measured the change in the beetles’ directions between two consecutive rolls when they rolled their balls out of an arena (1-m diameter) on a sunlit day or under a clear, moonlit sky (Fig. 1 B and C; control, ●). We tested the beetles’ orientation behavior at low sun elevations or (on full-moon nights) moon elevations (<30°). Under these conditions, a celestial body (sun or moon) and the celestial polarization pattern created by it are both clearly visible to the animals (Fig. 1A). In all cases, the change of direction between the two repetitions was clustered around 0° (P < 0.001 by V test, with an expected mean of 0°); that is, the beetles did not change their bearing between consecutive rolls (Fig. 1 and Figs. S1A and S2 A–D). This observation confirmed that both species have a consistent orientation behavior even at times when they are not naturally active.

Absolute change of direction in all behavioral experiments conducted in this study. Absolute changes of direction of the diurnal species (D; Upper) and absolute changes of direction of the nocturnal species (N; Lower) are shown. Error bars are circular SDs. (A) Absolute changes of direction of beetles (n = 20 per species) rolled twice under the sun (sun-sun) or when the sun was mirrored to the opposite sky hemisphere and the real sun was shaded (sun-mirrored sun) during one of their rolls. The same experiments were also conducted under a full-moon night (moon-moon, moon-mirrored-moon). In all cases, the beetles changed their bearing when the celestial body was reflected (P < 0.001 by Watson–Williams F tests), except when the nocturnal species rolled at night (P = 0.14, F = 2.18). The data are the same as shown in Fig. 1 B and C. (B) Absolute changes of directions of beetles of both species that were tested in an indoor arena using overhead polarized UV or green light as the only orientation cue. The polarizer either remained in place during both rolls (POL no turn) or was turned by 90° (POL 90°-turn). Purple bars show the absolute changes of direction under polarized UV light, and green bars show absolute changes of direction under polarized green light. Although both species followed the 90°-turn of the polarizer under UV light (P < 0.001 by Watson–Williams F tests), no change in rolling directions was observed under polarized green light (P > 0.05 by Watson–Williams F tests). The data are the same as shown in Fig. 2B and Fig. S4B. (C) Absolute changes of direction of beetles rolled three times under a celestial body (CB-CB-CB, diurnal species under the sun and nocturnal species under a full moon) and with respect to a green light spot as only orientation cue in an indoor arena (LS-LS-LS). Black bars show the absolute change of direction between the mean directions rolled under a celestial body and a green light spot (CBmean − LSmean). The data are the same as shown in Fig. 3C. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Permutation tests of all presented behavioral data (main text and Figs. 1 B and C, 2B, and 3C). Measured bearings were randomly permuted across individuals and experimental conditions. Histograms show the distribution of V values obtained from 1 million random permutations. Red lines indicate the unpermuted V values. ***P < 0.001; **P < 0.01. White panels show permutation tests of behavioral data from the diurnal species, and gray panels indicate permutation tests of behavioral data from the nocturnal species. Permutation of the control (Left) and test (Right) data when the diurnal species (A) and the nocturnal species (B) rolled twice with respect to the sun (Fig. 1B, control data) or when the sun was mirrored during the second roll (Fig. 1B, test data). Permutation tests of the control (Left) and test (Right) data when the diurnal species (C) and nocturnal species (D) rolled twice with respect to the moon (Fig. 1B, control data) or when the sun was mirrored during the second roll (Fig. 1B, test data). (E and F) Permutation tests of the polarized light data in the indoor arena. The beetles rolled twice under polarized UV light without a filter turn (Left) or when the filter was turned by 90° between two rolls (Right). (E) Permutation tests in the diurnal species (Fig. 2B, Left). (F) Permutation tests in the nocturnal species (Fig. 2B, Right). (G and H) Permutation tests between rolls under the sun or moon and with respect to a green light spot (Fig. 3B). (G) Permutation tests in the diurnal species (Fig. 3C, Left). (H) Permutation test of the V value in the nocturnal species (Fig. 3C, Right).

To investigate the relative importance of, or preference for, the two main celestial cues in the beetles’ compass system (sun or moon vs. polarized light), we also examined how beetles reacted when the two cues were set in conflict. Again, beetles were made to roll out from the center of the arena on two consecutive occasions: once in the light of the sun or the moon and once with the sun or full moon (celestial body elevations <30°) “displaced” to the opposite sky hemisphere using a mirror (at the start of either the first roll or the second roll), while simultaneously hiding the real sun or moon from the beetle’s view (Fig. 1 B and C; test, ○). If the beetle relies on polarized skylight as its main cue, it should adhere to its original rolling direction, which would still be visible (and unaltered) across the remainder of the visible sky. In contrast, if the beetle uses a celestial body (sun or moon) as its primary reference for orientation, it should use the reflected moon or sun as a directional cue and turn by about 180° with respect to its original rolling direction. We found that the result depended on which species was tested. Diurnal beetles followed the reflected sun or moon (i.e., they changed their rolling direction significantly toward the opposite sky hemisphere), both during the day (P < 0.001 by V test, with an expected mean of 180°) and at night (P = 0.002 by V test, with an expected mean of 180°) (Fig. 1 B and C, Left and Figs. S1A and S2 A and C). This result matches our previous findings (5, 17, 18) and suggests that diurnal dung beetles rely primarily on a celestial body for orientation; that is, they use either the sun or the moon as a compass. This cue preference was also true for the nocturnal species when orienting during the day [Fig. 1B, Right (P < 0.001 by V test, with an expected mean of 180°) and Figs. S1A and S2B]. However, when these nocturnal foragers rolled their balls in the light of the full moon, they displayed a different strategy: The beetles did not change their rolling direction (P = 0.996 by V test, with an expected mean of 180°), but kept rolling in their original direction of travel even when the moon was reflected back at them from the opposite direction [Fig. 1C, Right (P < 0.001 by V test, with an expected mean of 0°) and Figs. S1A and S2D]. In contrast to the diurnal species, the nocturnal species thus primarily followed the direction given by the skylight polarization pattern under dim light conditions. To confirm this observation, we also tested the nocturnal species rolling under a polarizing filter with a clear view of the full moon (elevation <30°) through the polarizer. The polarizer was placed on top of a screened arena, with the filter’s E-vectors either aligned (Fig. S3A) or in conflict (Fig. S3B) with the lunar polarized skylight. In contrast to the diurnal species, which orients with respect to the sun even if the polarizer is turned by 90° (5), the nocturnal species significantly changed its bearing in response to a change in the polarized light direction (Fig. S3). This experiment also shows that polarized light is ranked higher than the disk of the moon in the internal compass of nocturnal dung beetles. Overall, we show that despite the taxonomic proximity of both species, they rely on different celestial cues for their dominant directional reference under dim light conditions. Because both species exhibit similar orientation behaviors but differ in their activity window, our data suggest that nocturnal conditions (presumably the lower light intensity), or a circadian rhythm, cause the observed switch to a polarization compass in the nocturnal species.

To test if the polarization pattern dominates the orientation compass in the nocturnal beetle species, a UV-transparent polarization filter (HNP’B) with a diameter of 42 cm was placed above an arena (diameter of 60 cm). The filter was mounted on 10-cm legs, with a black cloth “skirt” attached to the edge of the holder (details are provided in ref. 5). During all trials, the moon was visible from within the arena. Each beetle rolled four times in total: once under the open sky, once under the filter with the E-vectors parallel to the main E-vector direction of the sky (A, control), once again under the open sky, and once under the filter with its E-vectors perpendicular to the main E-vector in the sky (B, test). The relative change of direction (Left, circles) and the absolute change of direction (Right, bars) are shown. Red lines indicate the mean change of direction, and red sectors indicate the circular SD. (A) Change of direction between rolls under the open sky or with E-vectors parallel to the main celestial E-vector (n = 20). The beetles’ rolling directions are clustered around 0°. (B) When the polarizer was turned by 90°, the beetles changed their heading by about 58°. This change of direction is significantly different from the change of direction observed in the control experiment (P < 0.001 by Watson–Williams F test).

Central Complex Cells Are Sensitive to Polarized UV Light.

Next, we sought to understand how the polarized light pattern and the position of the sun or moon are processed neurally in the beetle’s brain. To allow an accurate investigation of the neural substrate for polarization-dependent orientation, we first had to define at which wavelength of light the beetles perceive the skylight polarization pattern. Dacke et al. (15) suggested that dung beetles detect polarized light either in the green or UV wavelength. Therefore, we investigated the orientation performance of both of our model species in an indoor arena lit by polarized light from above, with a peak either in the UV (365 nm) or green (530 nm) wavelength (Fig. 2 A and B and Fig. S4). Each beetle was made to roll a ball twice from the center to the perimeter of a circular indoor arena. Between each roll, the polarizer either remained in place (control) or was turned by 90° (test). Under polarized UV light, both species changed their heading by 90° [Fig. 2B (in both species, P < 0.001 by V test, with an expected mean of 90°) and Figs. S1B and S2 E and F], whereas under polarized green light, the beetles were disoriented (Figs. S1B and S4). Thus, as in many other insects (19⇓⇓–22), both of our dung beetle species detect the skylight polarization pattern in the UV range.

Analysis of the polarization-dependent orientation systems in dung beetles. (A) Schematic drawing of the experimental design simulating the polarization pattern. The roll bearings of both species were analyzed with respect to zenithal polarized light in an indoor arena. (B) Response of diurnal (Left) and nocturnal (Right) dung beetles to polarized UV light. The angular difference between two rolls was recorded when the polarizer remained in place (control, black circles) or after a 90° turn of the polarizer (test, magenta circles). Both species oriented in response to the polarizer; that is, their changes of direction were clustered along the 0 to 180° axis in the control conditions (P < 0.001 by V test, n = 20 for each species) and oriented along the 90 to −90° axis when the polarizer was turned (P < 0.001 by V test, n = 20 for each species). The mean directions are indicated by gray (control) or magenta (test) lines, and error bars indicate circular SD. (C) Neural response of a TL neuron to a 360° revolution of a zenithal polarizer. (Middle) Firing activity of the neuron. (Upper) Sliding average of the tuning. (Lower) Bar indicates the rotation of the polarizer, and the disk shows a schematic drawing of the polarizer with E-vector orientation (double-headed arrows) at the initial point of rotation. (D) Neural tuning of four central complex cell types. The circular diagrams show the neurons’ firing activities plotted against stimulus orientation (in 10° bins). These plots are shown from the beetle’s point of view, with 0° in front of the animal. All neurons responded to the rotating polarizer [Rayleigh test: TL, P < 0.001 (nrotations = 2); CL1, P < 0.001 (nrotations = 10); TB1, P < 0.01 (nrotations = 10); CPU1, P < 0.01 (nrotations = 4)]. Error bars show SD. Black circles show background spiking activities. Red lines indicate the mean direction of the neuron.

(A) Schematic diagram of the experiment to compare orientation between diurnal and nocturnal dung beetles in green or UV light. Dung beetles of both species were tested in an indoor arena using an overhead polarized green light as the only orientation cue. (B) Response to polarized green light in diurnal (Left), and nocturnal (Right) dung beetle species. For each beetle, the angular difference between two consecutive rolls was recorded when the polarizer remained in place (control, black circles) or when the polarizer was turned by 90° (test, green circles). Only the diurnal species kept a constant rolling direction in the control condition (P < 0.001 by axial V test with an expected mean of 0°, n = 20). Directional changes in the nocturnal species were not clustered along the 0–180° axis (P = 0.84 by axial V test with an expected mean of 0°, n = 15). When the polarizer was turned by 90°, both beetle species were disoriented under polarized green light, as shown by the random distribution of green circles (diurnal species, P = 0.22 and nocturnal species, P = 0.28; both by axial V test, with an expected mean of 90°).

To analyze the tuning of the polarization-sensitive neurons, we presented the beetles with zenithal polarized UV light that had similar light intensities as in the behavioral experiments. Before starting to roll, dung beetles always climb on top of their balls and perform “orientation dances” in which they rotate around their own body axis (23). To simulate this vertical rotation underneath the natural polarization pattern, we rotated the polarizer by 360° while recording from compass neurons of the central complex. The dung beetle’s central complex consists of four brain areas, which have been described in detail in other insects (24): the upper and lower divisions of the central body (termed the fan-shaped and ellipsoid body in flies), the protocerebral bridge, and the paired noduli (Fig. S5 A–C).

Anatomy of the dung beetle brain and the central complex of the diurnal dung beetle species S. lamarcki. (A) Frontal view of a section through the dung beetle brain at the level of the central complex (CX). The brain is stained against the presynaptic vesicle protein synapsin. For simplification, only the left optic lobe (OL) is shown. The 3D maximum intensity visualization of the central complex in the frontal view is shown in red. (B) Frontal view of a 3D reconstruction of all subdivisions of the same central complex as shown in A. The central complex consists of four brain areas: the lower (CBL) and upper (CBU) divisions of the central body, the noduli (No), and the protocerebral bridge (PB). (C) Oblique view scaled to the same size as B shows the relative position of the neuropils of the central complex. (D) Three-dimensional reconstructions of the central complex cells analyzed and their innervated brain areas (transparent). TL and CL1 neurons of the lower division of the central body form the early processing stage of polarization signals, whereas tangential neurons of the protocerebral bridge (TB1) and columnar cells of the protocerebral bridge and upper division of the central body (CPU1) form the late processing stage of polarization signals in the central complex. CBL, lower division of the central body; CBU, upper division of the central body; d, dorsal; m, median; p, posterior; PB, protocerebral bridge. [Scale bars: A, 500 μm (whole brain); B–D, 100 μm.]

Using single-cell tracer injections, we were able to identify (and record from) all four types of central complex neurons known to process polarization signals in other orienting insects (14) (Fig. S5D). According to a proposed processing network in the locust’s central complex (25), these cells can be categorized into early and late processing-stage neurons: Input tangential (TL) neurons and columnar (CL1) neurons represent the early processing stage, whereas tangential (TB1) cells and columnar (CPU1) neurons represent the late processing stage (Fig. S5D). In total, we recorded from 21 central complex neurons [TL: n = 8 (three diurnal, five nocturnal), CL1: n = 9 (four diurnal, five nocturnal); TB1: n = 3 (one diurnal, two nocturnal); CPU1: n = 1 (nocturnal)] while rotating the polarizer by 360° above each beetle at least twice (one clockwise rotation and one counterclockwise rotation) (Dataset S1).

Neurons of the same type showed similar responses, independent of the species’ identity. When stimulating early processing-stage cells (TL and CL1) with a rotating polarized UV light, the neurons were maximally excited at a particular orientation of the polarizer and were maximally inhibited at the perpendicular orientation (Fig. 2 C and D), demonstrating their angular sensitivity to polarized UV light. In contrast, late processing-stage cells (TB1 and CPU1) typically showed a firing activity that was similarly, but less strongly, modulated during polarizer rotation (Fig. 2D and Fig. S6 A and B). This neural tuning is well in line with observations from the locust’s central complex and can most likely be explained by a multimodal function of the late processing cells in the central complex network (14, 25). In summary, the dung beetle’s central complex acts as a key processing center for polarized light analysis. However, to function as a robust internal compass for straight-line orientation, it would also have to identify and integrate the azimuthal position of the sun (or moon), as has recently been shown for locusts and monarch butterflies (26, 27).

General characteristics of compass neurons in the beetle’s central complex. (A) Modulation depth (M) was calculated to define the strength of the modulation of the cells without any stimulus presentation (in darkness, black bars) and when stimulated with a rotating polarizer (purple bars) or a moving green light spot (green bars). Figures show the modulation depths of all analyzed neurons (numbers) and the average (A) modulation depths. From left to right: M values and average M values for eight TL neurons, M values and average M values for nine CL1 neurons, M values and average M values for three TB1 neurons, M values for one CPU1 neuron. TL and CL1 neurons are significantly more strongly modulated during stimulus presentation than without any stimulation. (B) Ratio between Mstimulus and Mdarkness contains information about how much each type of neuron was modulated during stimulus presentation (purple bars, rotating polarizer, UV light; green bars, moving green light spot) with respect to the background modulation. (C) Comparison between the modulation depth in bright light and dim light. This relationship describes the strength of the neural response during dim light conditions compared with the response at high light intensities (1.0). Neurons in the nocturnal species (Left, n = 5) and neurons in the diurnal species (Right, n = 6) are shown. Whereas the response to the green light spot dropped to about 40% in nocturnal species, the neural response to polarized UV light was reduced to about 40% in the diurnal species. (D) Preferred directions [Φmax(POL), mean directions] to polarized UV light of all analyzed 21 central complex neurons. (E) Preferred azimuth directions [Φmax(Lightspot), mean directions] to the moving green light spot of 19 central complex neurons that showed a significant neural response. (F) Absolute angular differences between the preferred polarized UV directions and the green azimuth directions. In A–C, the error bars represent the SD.

Central Complex Neurons also Encode the Sun/Moon Azimuth.

During our experiments, we also analyzed how the very same neurons that responded to polarized light were modulated in response to a moving unpolarized green light stimulus. In honey bees, a green light spot is interpreted as the sun’s direction (28⇓–30). To test whether the sun or moon can be replaced by an artificial green light spot also in the compass system of the beetles, we let the beetles roll first under the clear sky and then again immediately afterward in an indoor arena, lit only by a green light spot (or vice versa, using the light spot first followed by the celestial cue) (Fig. 3 A and B). Interestingly, even when the beetles had a green light spot as their sole orientation cue, their orientation was equally precise (nocturnal species) or only slightly less precise (diurnal species) than their performance outdoors when many celestial cues were available (Fig. S1C). We found that irrespective of whether the beetles (n = 20 diurnal beetles and n = 20 nocturnal beetles) first rolled under the green light spot (elevation about 25°) or under the sun or moon, the beetles’ heading was maintained in both situations (i.e., the change of direction was clustered around 0°) (Fig. 3C; in both species, P < 0.001 by V test, with an expected mean of 0°; Fig. S2 G and H). Both dung beetle species thus clearly orient in a similar way to a green light spot as they do to a celestial disk.

Analysis of the azimuth-dependent orientation systems in dung beetles. (A) Schematic illustration of the experimental setup. The roll bearings of beetles were analyzed with respect to a green light spot in an indoor arena and compared with the rolling directions with respect to the sun (diurnal species) or moon (nocturnal species) outdoors. The elevation of the light spot and the celestial body was similar (<30°). (B) Beetles rolled their balls three times under a celestial body (yellow dots) and three times with a light spot (green dots) as their only orientation cue. The angular difference between the mean direction relative to the celestial body (yellow line) and the light spot (green line) was measured as the change of direction (black sector). (C) In diurnal (Left, n = 20) and nocturnal (Right, n = 20) beetles, the change of direction was clustered around 0° [P < 0.001 by V test; μdiurnal (±SD) = −24.5° ± 46.25°; μnocturnal = −4.6° ± 37.23°]. Mean direction is shown as a green line, and error bars show SD. (D) Neural activity of a CL1 cell to a green light spot moved on a circular path around the beetle’s head (elevation of 30°). (Middle) Firing activity of the neuron. (Upper) Sliding average of the tuning. (Lower) Solid line shows the position of the light spot with respect to the beetle’s head, and the schematic drawing shows the position of the light spot at the initial position relative to the animal. (E) Firing activity of three central complex cells (recorded in both species) to a light spot. The circular plots show the neuron’s firing activity plotted against stimulus orientation (in 10° bins). All plots are shown from the beetle’s point of view, with 0° in front of the animal. The cells respond significantly to the green light spot [Rayleigh tests: TL; P < 0.01 (nrotations = 2); CL1, P < 0.001 (nrotations = 4); TB1, P < 0.001 (nrotations = 2)]. Error bars show SD. Black circles show background firing activities. Red lines indicate the mean direction of the neuron.

Next, we simulated beetles rotating under the sun or the moon (but in the absence of a celestial polarization pattern) and analyzed the response of 20 neurons to a moving green light spot that moved on a circular path around the beetle’s head. In total, 19 (TL: n = 7, CL1: n = 9, TB1: n = 3) of the polarization-sensitive central complex neurons recorded above showed a significant response also to the moving green light spot (Fig. 3 D and E; P < 0.05 by Rayleigh test) and were clearly modulated by the stimulus position (Fig. S6 A and B). Early processing-stage cells (TL and CL1) were excited by the green light spot when it passed a certain position in the visual field. TL neurons showed an excitation with a narrow receptive field. In contrast, CL1 neurons showed an additional strong inhibition in the opposite visual field (Fig. 3E). Two CL1 neurons showed only inhibition as a response to the green light spot. In contrast, the response characteristics of late processing-stage (TB1) cells were diverse but always featured an increased average neural activity at a certain azimuth of the light spot (Fig. 3E). In summary, our data show that compass neurons in the beetle’s central complex encode the pattern of polarized skylight and are suited to encode the sun’s or moon’s azimuth (especially cells at an early processing stage). In combination, these two cues can be used to generate a robust internal sky compass signal for straight-line orientation. Next, to test the relative representations of both signals at the neural level, we set out to analyze the neural activity of central complex cells when the animal was exposed to both stimuli simultaneously.

Behaviorally, we found that diurnal dung beetles primarily use a celestial body for orientation, both during the day and at night. Nocturnal dung beetles also use this cue under daylight intensities but switch to a polarization compass for nocturnal orientation (Fig. 1). To analyze how this difference is encoded in the central complex of the beetle, we simultaneously presented the dung beetles with dorsal polarized UV light and an unpolarized green light spot while recording from early-stage compass cells (the same TL and CL1 cells as above). To imitate the natural polarization pattern of the sky, the electric field vectors of the zenithal polarizer were oriented perpendicular to the light spot’s azimuth, and the light intensity was adjusted to match either the conditions at low sun elevations or the conditions at low moon elevations. Because dung beetles seem to use a pure celestial compass system, without landmark information (8), this setup approximates the situation the orienting beetles would normally experience at low sun or moon elevations (Fig. 1A). When presenting both stimuli simultaneously to a TL neuron at brighter or lower light intensities corresponding to a low sun or moon elevation, the neuron of the diurnal beetle (Fig. S7A) ignored the polarization signals and responded exclusively to the green light spot. This neural response was also true for a TL neuron of the nocturnal beetle at brighter light intensities (Fig. 4A). However, when exposed to dimmer light intensities matching full moonlight, the TL neuron of the nocturnal species no longer responded to the artificial celestial body. Instead, the firing activity of the neuron was clearly modulated by the polarization channel (Fig. 4A).

(A) Encoding the celestial cue preference in the central complex of diurnal dung beetles. (Upper) Sliding average of the response of a TL neuron to a rotating polarizer (UV light) (Left, magenta), a moving green light spot (Middle, green), and when both stimuli were shown simultaneously at high light intensities (Right, black). Gray curves indicate individual rotations. (Lower) Moving sliding average of the response of the same neuron, with light intensities that simulated conditions of a full-moon–lit night. In contrast to TL neurons in the nocturnal species, the diurnal neuron always responded to the simulated celestial body independent of the light intensities. Solid lines show the stimulus timing; disks and circles indicate stimulus type [polarized light (magenta) and or light spot (green)]. imp, impulses. (B) Weights c of the neural response to different light stimuli of TL (blue circles) and CL1 (red circles) cells. (Left) Neural responses of central complex neurons (TL and CL1) of the nocturnal species to polarized UV light, a green light spot, or both stimuli presented simultaneously. The white background denotes high light levels, and the gray background shows dim light levels. Numbers show the number of cells analyzed. (Right) Same, but for central complex cells analyzed in the diurnal species. The data are the same as shown in Fig. 4C.

To quantify which of the two stimuli dominates the neural responses under the two different light conditions, we combined a Gaussian fit (describing the response to the light spot) and a sine-square fit (describing the neural response to polarized light) in a single weighted fit function. This curve fit method enabled us to obtain a weighting factor between 0 and 1. A weighting factor between 0 and 0.5 indicates that the neuron responded with a 360° periodicity to the light stimulus (i.e., to the sun or moon azimuth), whereas a weighting factor between 0.5 and 1 indicates that the cells responded with a 180° periodicity (i.e., to the polarized light) (Fig. 4B). The results of this quantitative analysis matched exactly what we observed in the behavior of the beetles: When stimulated exclusively with polarized UV light, the spiking activity of central compass neurons was sinusoidally modulated, with a high average weighting factor of 0.79 ± 0.12 (mean ± SD) and 0.87 ± 0.06, no matter whether we tested diurnal or nocturnal beetles and irrespective of light intensity (Fig. 4C and Fig. S7B). Conversely, when we showed only a green light spot to the animal, the weighting factor was relatively low, again irrespective of species or light condition (Fig. 4C, between 0.21 ± 0.14 and 0.39 ± 0.08). When we stimulated with both cues simultaneously, the results depended upon the species. In the diurnal species, at both light intensities, the cells’ firing activity was modulated similar to the firing activity observed with the green light spot alone, suggesting that central complex neurons encode the sun or moon as the main celestial cue. In the nocturnal species, in contrast, this weighting was only the case at high light intensities, indicating that these neurons encode the sun’s position (Fig. 4C). However, when shown both cues at low light intensities (simulating a clear sky lit by a full moon), the firing activity of neurons in the nocturnal species was similar to the firing activity observed with polarized light stimulation alone (weighting factor: 0.75 ± 0.11; n = 5) showing that the neurons preferentially encode polarized skylight at this dimmer light level.

In summary, the neural encoding of celestial orientation cues in bright and dim light conditions closely matches the observed cue preference of these cues during the natural orientation behavior of our model species in the field (Fig. 5): Nocturnal dung beetles use the celestial polarization pattern as their main compass cue at night, whereas diurnal beetles use the sun as their main compass cue during the day.

Summary of results. The illustration of the ball-rolling dung beetle represents the behavioral experiments. The schematic drawing of the cell with the recording trace represents the physiological experiments. Diurnal species (Left) and nocturnal species (Right) are shown. Both were analyzed at low sun elevation (behavior) or with light conditions simulating low sun elevation (physiology) (Upper) and at low moon elevations (behavior) or with light simulating low moon elevation (physiology) (Lower). The diurnal species uses the celestial body as its primary orientation cue during both day and night, but the nocturnal species switches to the polarization pattern as its primary cue when rolling balls at night. The behavioral demonstration of the compass cue preference accurately matches the neural encoding of the celestial cue weighting in the compass neurons of the brain. In each panel, pale gray cues indicate that this cue is not used as the main reference in this condition.

What then are the mechanisms that generate the switch to polarized light under dim light conditions in the nocturnal species? Studies in locusts and crickets have shown that the tuning of compass neurons to polarized light is intensity-independent above a particular threshold level of polarized light intensity (31, 32), whereas responses to unpolarized light strongly depend on light intensity (32). Consequently, when both stimuli are dimmed to lunar light intensities, only the response in the channel that is processed in an intensity-dependent manner should be diminished (i.e., the input from the light spot in these experiments). At lunar light intensities, the inputs should thus be modified in favor of the polarization channel for as long as the polarization analyzer of the eye is sensitive enough to detect a signal. In the nocturnal dung beetle, we found that the neurons responded reliably to bright as well as dim polarized light. The neuronal response in the diurnal species, on the other hand, was weak or absent at dim light intensities compared with the neuronal response at high light intensities (Fig. S6C). These neural responses might, at least partially, be a result of the size ratio between the dorsal rim area and main retina. The dorsal rim areas of crepuscular beetles are enormous (33), and our first preliminary observations suggest that this is also true for the nocturnal species tested here. In contrast, the dorsal rim areas of the diurnal species seem to be relatively small. The higher sensitivity to polarized light, and thus the weighting of different celestial cues in central complex neurons, might therefore be determined at an earlier stage in the brain. This difference in sensitivity to the polarization of light is also reflected in the orientation behavior of our two model species: Under dim light intensities, nocturnal navigators will adhere to the direction given by the polarization pattern rather than to the direction indicated by the moon’s position, whereas the diurnal species follows the direction derived from the moon (Fig. 5). This cue preference raises an important question regarding the advantages that a sky-wide polarization pattern offers over a bright point source (in the form of a moon) for nocturnal orientation. Because this difference in cue preference is a phenomenon observed only under dim light intensities, the answer is most likely a gain in sensitivity. One excellent strategy to gain visual sensitivity is to pool signals from many photoreceptors in one second-order neuron, a mechanism known as spatial summation (34, 35). The polarization pattern extends over the entire sky, and therefore allows the receiver to integrate signals over a wide visual field. At low light intensities, the polarization pattern should be a more reliable orientation cue than a moon, appearing as a dimly illuminated disk in the sky that may also be blocked from view by clouds. Moreover, even under the low light of a quarter or crescent moon, S. satyrus can consistently roll a straight-line path when the moon itself is blocked from view, almost certainly using the lunar polarization pattern for orientation (9). Similarly, crickets are able to detect polarized light even at light intensities equivalent to the light intensities of a moonless night sky (36). Under all these extremely dim conditions, spatial summation might be even more relevant for the orientation compass of nocturnal insects, as hypothesized by Labhart et al. (31); thus, the polarization pattern might offer the most reliable celestial reference for nocturnal navigation. Even though the moon, in any phase, is only available for about half of the month, it can still polarize the night sky when it is up to 15° below the horizon (37), extending the period when S. satyrus can reliably forage. Nevertheless, when the sun and moon are further below the horizon than 15°, and the night sky has no overall pattern of polarization, S. satyrus can fall back on the stars of the Milky Way to roll dung balls through the African bush (16), a cue that presumably ranks lowest in the cue preference of orientation signals.

In summary, our study clearly demonstrates how the visual ecology of an orienting animal is reflected in its orientation compass system. Here, the dynamic activity of neurons in the central complex provides the nocturnal orienting animal with a simple yet efficient tool to lock onto the most reliable signal available. This dynamic tuning generates a robust internal compass that can ensure precise orientation behavior with the cues available.

Materials and Methods

General.

Investigation of the Celestial Cue Preference.

To test which of the cues (sun/moon or polarization pattern) dominates the sky compass of dung beetles, we analyzed the rolling behavior with the celestial cues aligned or set in conflict (SI Materials and Methods).

Indoor Experiments with Polarized Light.

To analyze the wavelength range over which the beetles perceive polarized light, we presented zenithal polarized light in the UV or green range to the beetles in an indoor arena (SI Materials and Methods).

Orientation Under the Sun and a Green Light Spot.

To test whether beetles interpret a green light spot as the sun or moon, we compared the bearings taken with respect to the sun or moon with the bearings taken with respect to a green light spot indoors. Details are provided in SI Materials and Methods.

Physiology.

To analyze neural activity, we used a standard electrophysiology method to record responses of neurons intracellularly. Details are provided in SI Materials and Methods.

Visual Stimulations.

To test whether central complex neurons are sensitive to polarized UV light and/or to a green light spot, we designed a stimulus similar to the one described by Pfeiffer and Homberg (38). Details are provided in SI Materials and Methods.

Anatomy.

Immunohistochemistry, image acquisition, and 3D reconstruction procedures for neurons and neuropils have been described in detail by el Jundi et al. (39). Details are provided in SI Materials and Methods.

Data Analysis.

All experiments were analyzed with custom-written programs in MATLAB (MathWorks). Details are provided in SI Materials and Methods.

SI Materials and Methods

General.

All experiments were performed with adult dung beetles of the diurnal species S. (Kheper) lamarcki and the nocturnal species S. satyrus (Coleoptera; Scarabaeidae). Both species were collected in South Africa using baited pit-fall traps. They were kept outdoors in soil-filled plastic bins (30 × 22 × 22 cm) and were fed fresh cow dung daily. A net prevented the beetles from escaping but still allowed them to see the full sky at all hours of the day. Behavioral experiments were conducted in the beetles’ natural habitat on the game farm “Stonehenge” in North West province, South Africa (24.32°E, 26.39°S). Experiments were performed in January and November 2013 and 2014 at sun and moon elevations between 15° and 30°. Under these conditions, both the polarization pattern and the sun or moon are clearly visible to the animals.

For anatomical and electrophysiological studies, dung beetles of both species were transferred to Sweden, where they were housed in large boxes (50 × 36 × 27 cm) at the Department of Biology (Lund University). The day-active species was kept under a 12-h light/dark cycle, with lights on at 10:00 AM and at a room temperature of 26 °C. The nocturnal species was kept in a second animal room (12-h light/dark cycle), with an 11-h shifted light/dark cycle (lights off at 11:00 AM). These light/dark cycles allowed an easy analysis of the neural activity of the diurnal species during its subjective day and of the nocturnal species during its subjective night. Both species were fed with fresh cow dung collected from a farm close to Lund. Under these conditions, the beetles survived for up to 1 y.

Behavior.

Investigation of the celestial cue preference.

To test which of the celestial cues (sun/moon or celestial polarization pattern) dominates the sky compass of dung beetles, we analyzed the rolling behavior of both of our model species with the celestial cues aligned or set in conflict. Forty beetles (20 diurnal beetles and 20 nocturnal beetles) were tested during the day (sun elevations <30°, in the afternoons), and 40 beetles (20 diurnal beetles and 20 nocturnal beetles) were tested under a full-moon night (moon elevation <30°, at the beginning of the nights). Individual beetles were released to the south of their dung balls in the center of a leveled circular wooden arena (1-m diameter), coated with a thin layer of soil. After release, the beetles immediately climbed up onto their ball and started to roll it in a certain direction. As soon as a beetle reached the perimeter of the arena, the bearing was recorded and the beetle was returned with its ball to the center of the arena to repeat the experiment. Each beetle performed three rolls: twice with a full sky visible, including the sun or moon (control), and once with a full sky visible and with the sun or moon displaced 180° (test). The latter was achieved with a mirror (20 × 20 cm, ∼1.5 m away from the beetle) that reflected the sun/moon from an opposite azimuth to the actual sun or moon azimuth while hiding the real sun or moon with a square board (100 × 75 cm, ∼1.5 m away from the beetle). Half of the beetles experienced the mirrored sun or moon first, and half of the beetles experienced the nonmanipulated sky first. The order in which this testing was done was randomized.

Indoor experiments with polarized light.

Previous studies suggest that dung beetles detect polarized light in either the green or UV range (15). To analyze the wavelength range over which the present model species perceive polarized skylight, we presented zenithal polarized light in either the UV or green range to the beetles in an indoor arena that we constructed at the field site in South Africa. The light source consisted of six high-power UV light-emitting diodes (LEDs; emission peak at about 365 nm; LZ1-00U600, LedEngin, Inc.) and six white LEDs (X42182; Seoul Semiconductor), which were mounted on a large aluminum plate. Both UV- and white LEDs were arranged in a circle to avoid providing an irregular pattern that could be used by the beetles for orientation. The plate was suspended 75 cm above the center of a circular wooden arena (similar to the outdoor arena, but with a diameter of 60 cm). A thick black curtain hanging down from the edge of the LED plate blocked any visual cues from outside the arena. A UV-transparent polarization filter (HNP'B, Polaroid) with a diameter of 42 cm was placed under the lights, above the center of the arena. The filter was mounted on a filter holder with 14-cm-high legs. Similar to previous experiments, a black cloth was attached to the edge of the filter holder (5) so that the beetles were only able to see light that passed through the dorsal polarizer. We produced polarized green light by turning on the white-light LEDs and adding a green filter to the filter holder. The green filter was mounted on top of the polarizer and restricted the light stimulus to a wavelength range of 480–580 nm (with a peak at 530 nm). The intensities of polarized UV and green light were each adjusted to a photon flux of about 2.2–2.9 × 1013 photons per square centimeter per second using neutral density filters (LEE Filters) placed on top of the polarizer.

Twenty beetles of each species were individually released underneath the polarizer in the center of the arena and were allowed to roll their balls. With a few exceptions, each beetle rolled its ball a total of six times: three times under UV light and three times under polarized green light. Half of the beetles rolled under green light first, and the other half rolled under UV light first. The rolling behavior of the beetles was observed from above using a video camera (VP-HMX20C; Samsung) mounted with the lens in an aperture in the center of the LED plate. Once a beetle reached the edge of the arena, the rolling direction was noted and the beetle was returned to the center of the arena. Before the second roll, the filter either remained in place (control) or was turned by 90° (test). To measure the orientation behavior of the beetles under both conditions, we calculated the angular difference between two consecutive rolls without a filter turn (control) and when the filter had been turned by 90° between two consecutive rolls (test). Experiments with the diurnal species were conducted during the day, whereas the orientation behavior of the nocturnal species was tested at night.

Orientation under the sun and a green light spot.

To test whether beetles use a light spot as an orientation cue in a similar way to the sun or moon, we compared the bearings between two consecutive rolls under the natural sun and under a green light spot. Each beetle rolled its ball out of the center of an arena six times. Three of these rolls were performed under the natural sky with the sun (diurnal species) or the moon (nocturnal species) as an orientation signal, and three rolls were performed under laboratory conditions in the indoor arena with only a green light spot as a reference. The green light spot consisted of a single green LED (peak at about 530 nm; LZ1-00G100, LedEngin, Inc.) that was mounted on a tripod and positioned close to the perimeter of the indoor arena, ∼45 cm away from the center of the arena at an elevation of about 20–30°. During all experiments with the diurnal and nocturnal species, the green light spot had an intensity of about 3.5 × 1013 photons per square centimeter per second. The azimuth of the green light spot was always shifted by 90° relative to the azimuth of the sun or moon to ensure that the beetles were not using magnetic or other nonvisual cues. Half of the beetles rolled under the natural sky first, whereas the other half experienced the green light spot first. For each beetle, we calculated the angular difference (change of direction) between the mean directions of the three rolls under the full visible sky and the mean directions of the three rolls measured relative to the green light spot.

Physiology.

Preparation.

To analyze the neural activity of central complex cells, we used a standard electrophysiology method to record responses of individual neurons intracellularly, using sharp electrodes. According to their different diel activity, neurons of the diurnal species were investigated during their subjective day, whereas neurons of the nocturnal species (with two exceptions) were recorded during their subjective night. Accordingly, most of the nocturnal beetles were dissected under dim red-light conditions. To prepare the animals for the recordings, the legs were removed and the stumps and mouthparts were sealed with wax. Afterward, the beetles were fixed to a horizontal holder, a small part of the distal thorax was removed, and the dorsal neck muscles were cut. The removal of these muscles enabled an overstretching of the animal’s neck and the fixation of the head to the holder so that dorsal regions of the eyes were oriented anteriodorsally. The head capsule was then opened posteriorly, and the trachea and muscles above the central brain were removed. Finally, to facilitate electrode penetration, the neural sheath above the posterior central brain was removed. A silver wire inserted into the opened thorax served as the reference electrode.

For intracellular recordings, sharp electrodes (resistance: 60–120 MΩ) were drawn from borosilicate capillaries (inner diameter of 0.75 mm, outer diameter of 1.5 mm; Hilgenberg) using a Flaming/Brown horizontal puller (P-1000; Sutter). Electrode tips were filled with 4% neurobiotin (Vector Laboratories) (3 mg) dissolved in 1 M KCl (75 μL), and the electrode shanks were filled with 1 M KCl. Neural signals were amplified (magnification of 10×) with a BA-03X amplifier (NPI). The signals were audio-monitored (HP2.1 Compact Speaker System; Hewlett Packard) and visualized using a digital oscilloscope (PM3302; Philips). A digitizer (1401-3; Cambridge Electronic Design) was used to sample the signals at a rate of 5 kHz. The sampled signals were stored on a computer using Spike2 software (version 7.08; Cambridge Electronic Design). After visual stimulation, neurobiotin was injected into the neuron using a constant positive current of about 2–3 nA for 1–4 min.

Visual stimulations.

To test whether central complex neurons are sensitive to polarized UV light and/or to an unpolarized green light spot, we designed a stimulus similar to the one described by Pfeiffer and Homberg (38), but with modifications that allowed us to monitor the neural activity to both stimuli simultaneously. Zenithal polarized light was produced by passing the light of a UV LED (emission peak at 365 nm; LZ1-00U600, LedEngin, Inc.) through a UV-permeable polarizer (HNP’B, 2-cm polarizer diameter). To increase the size of the light stimulus, we added a concave lens and a diffuser (quarter white diffusion; LEE Filters) between the UV LED and the polarizer. This setup resulted in an angular extent of the stimulus at the beetle’s eye of ∼5.4°. The polarizer was mounted on a rotation stage (DT-50; Micos) that allowed the rotation of the polarizer in both clockwise (0–360°) and anticlockwise (360–0°) directions with an angular velocity of 60° per second. In addition, an unpolarized green light LED (emission peak at 530 nm; LZ1-00G100, LedEngin, Inc.) was mounted on the head of an aluminum arm (with a flexible head). This arm was attached to the rotation stage, perpendicularly to the 0–180° axis of the polarizer. The head of the arm was adjusted so that the green LED moved on a circular path around the center of the beetle’s head at an elevation of about 30°. The angular size of the green light spot was ∼3.8°, and the movement velocity was 60° per second. To match the light intensity conditions to the light intensity conditions at the night/daylight sky (low sun/moon elevations), we measured the light intensity of the polarized UV light and green light spot by placing the sensor of a spectrometer (USB 2000; Oceans Optics) exactly in the position and orientation in which the animals would be facing during experimental stimulation. The total photon fluxes of the UV and green LEDs were determined by calculating the integral underneath the emission spectra obtained. They were then compared with the light intensity of the day sky and night sky in the same range of wavelengths (40). Accordingly, the photon flux for both polarized UV and unpolarized green light stimuli was adjusted to about 8.2–8.5 × 1013 photons per square centimeter per second. To mimic the moon, we used a similar arm to the one described above, but mounted opposite to the first arm. The light intensity of this green light spot was adjusted to about 8.1–9.9 × 1010 photons per square centimeter per second to simulate the light intensity at low moon elevations [about 7.5 ×1010 photons per square centimeter per second (40)]. Similarly, the light intensity of the zenithal polarized light was adjusted to the same light intensity by inserting neutral density filters between the UV LED and the polarizer.

Anatomy.

Tracer injections.

After injection of neurobiotin into the neurons, brains were dissected out of the head and fixated in a solution containing 4% paraformaldehyde (4 mL), 0.25% glutaraldehyde (0.25 mL), and 2% saturated picric acid (2 mL) in 0.1 M phosphate buffer (93.75 mL) overnight at 4 °C. The brains were then washed for 1 h in 0.1 M PBS (pH 7.4) and incubated for 3 d with streptavidin conjugated to Cy3 (1:1,000 ratio; Cy3-streptavidin, Dianova) in 0.1 M PBS containing 0.3% Triton X-100 (PBT). Afterward, the samples were washed four times in 0.1 M PBT and dehydrated in an ascending ethanol series (25–100%, 15 min each). After an ethanol/methyl salicylate (1:1 ratio, 15 min) step, brains were cleared in methyl salicylate for at least 35 min and mounted in Permount (Fisher Scientific) between two coverslips. To prevent compression of the brains, six reinforcement rings were used as spacers.

Image acquisition and 3D reconstructions.

Brains were scanned for stained neurons with a laser scanning microscope (LSM 510 Meta; Zeiss) using an objective with a magnification of 25× (LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27; Zeiss). Cy3 signal was scanned using a 543-nm HeNe laser. All neurons were scanned in several image stacks with a resolution of 1,024 × 1,024 (voxel size: 0.1–0.5 × 0.1–0.5 × 0.5–1.5 μm). The image stacks obtained were processed on a personal computer using Amira 5.3.3 software (FEI, Visualization Sciences Group). The 3D reconstruction procedure for neurons and neuropils has been described in detail (39).

Data analysis.

Behavioral data.

Behavioral experiments were analyzed in MATLAB (MathWorks). Changes of direction were calculated by measuring the angular difference between two rolls. The distribution of the changes of direction was tested using a unimodal V test (6) with an expected mean of 0° for control data and 180° for reflected sun/moon experiments, or a bimodal V test with an expected mean of 0/180° for controls and 90/270° for test data in the polarization experiments. To test the reliability of the V test, all data were analyzed using permutation tests (Fig. S2). The measured angles were randomly permuted (1 million times) across individuals, and experimental conditions and the corresponding V value were calculated. The significance of the experiment was judged by calculating the percentage of permutations that resulted in a V value greater or equal to the V value calculated from the unpermuted data.

Physiological data.

Spike trains were exported from Spike2 and evaluated by a custom-designed script in MATLAB. First, all spikes were detected using threshold-based event detection. All events during a 360° rotation of the polarizer or moving green light spot were assigned a particular angle relative to the polarizer or light spot. The distribution of these angles was then analyzed statistically for significant difference from uniformity using the Rayleigh test (41). A neuron was defined as polarization-sensitive if P < 0.05 for a bimodal test or azimuth-sensitive if P < 0.05 for a unimodal test. We considered only stimulation periods at high light intensities because the neural responses at low light intensities were sometimes marginal or absent, and thus inadequate as a criterion for polarization or azimuth sensitivity. If a neuron was polarization- and/or azimuth-sensitive, the corresponding mean vector of the distribution was used to define the preferred orientation for polarized light [Φmax(POL)] or as the preferred azimuthal direction [Φmax(lightspot)] of the neuron (Fig. S6 D and E). The angular relationship between polarized light and azimuthal direction of a neuron was defined by the absolute difference between the corresponding preferred directions [|Φmax(POL) − Φmax(lightspot)|] (Fig. S6F).

To analyze the neural responses quantitatively, we calculated the response modulation depth (M) of the cells during stimulus presentation (Fig. S6 A–C). The stimulation periods were divided into 18 bins (each 20° wide), and M was defined using the following equation (42):M=∑i=118|ni−n¯|,[S1]where ni is the spike rate (in spikes per second) in bin i and n¯ is the mean spike rate over the whole stimulation period. M contains information about how strongly the neurons were modulated during stimulus presentation (higher M values indicate a stronger modulation). In addition, to measure how strongly the neurons were modulated relative to the background spiking activity, we calculated the M values during periods (with two exceptions, the period was always 6 s) without stimulation (Mdarkness). The modulation depths of each type of neuron were statistically compared with the corresponding Mdarkness using the Wilcoxon signed-rank test.

To analyze which light source (polarizer or green light spot) dominated the neural response of the cells when both stimuli were presented to the animal, we combined a sine-square fit (Eq. S2, describing the neural response to polarized light) and a Gaussian fit (Eq. S3, describing the response to the green light spot) in a single weighted fit function (Eq. S4):f1(x)=A1⋅(sin(x−w1))2,[S2]f2(x)=A2⋅e−(x−w2)2/p,[S3]f(x)=c⋅f1+(1−c)⋅f2,[S4]where A1 and A2 are the amplitudes and w1 and w2 are the phase shifts of the sine-square and Gaussian fit functions, respectively. The width of the Gaussian fit is controlled by p, and it was limited in a way that restricted the width of the Gaussian function (FWHM) to between 90° and 180°. The weighting factor (c) defines to which amount each of the functions is used to describe the neural response. For c values between 0 and 0.5, the neural response was more closely described by Eq. S3 (responses to the light spot), whereas c values between 0.5 and 1 indicated that the neuron’s response more closely resembled Eq. S2, suggesting that the cell responded to the polarized light stimulus.

Acknowledgments

We thank Drs. James Foster, Stanley Heinze, Octavian Knoll, Keram Pfeiffer, and Christine Scholtyssek for their helpful discussions and comments. We are grateful to Ted and Winnie Harvey for their invaluable help in the field. We also thank Drs. Erich Buchner and Christian Wegener for providing the antisynapsin antibody. Funding for this project was provided by Vetenskapsrådet, the Wallenberg Foundation, the Wenner-Gren-Foundation, the Royal Physiographic Society in Lund, the Lars-Hierta Memorial Foundation, and the South African National Research Foundation.

(2001) Polarization of the moonlit clear night sky measured by full-sky imaging polarimetry at full Moon: Comparison of the polarization of the moonlit and sunlit skies. J Geophys Res106(D19):22647–22653

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